The Paracaspase MALT1 Acts Independently of Pre-B-Cell Receptor Signaling As a Key Factor in Leukemic Cell Survival in Precursor B-Cell Acute Lymphoblastic Leukemia

Despite a central role for B-cell receptor precursor (pre-BCR) pathway in precursor B-cell acute lymphoblastic leukemia (B-ALL), there is limited available data on therapies that aim to disrupt this pathway. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for BCR-mediated NF-κB activation. We recently showed that targeting MALT1 with the small molecule inhibitor MI2 is effective in CLL, including drug-resistant clones (Saba Can Res 2017). We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of inhibiting its activity. First, we tested MALT1 expression by immunoblot in B-ALL using 17 cell lines representing the disease spectrum (7 pro-B: REH, SEMK2, TOM1, RS4;11, NALM21, Z119, BV173; 8 pre-B: HB11;19, NALM6, RCH-ACV, SMS-SB, 697, KASUMI2, KOPN8, HPB-NULL; and 2 mature/Burkitt: 2F7, RAJI), and found that MALT1 was expressed in all cell lines at different levels. To determine sensitivity to MALT1 inhibition we used two molecules: Z-VRPR-fmk, a highly selective MALT1 blocking peptide, and MI2, a small molecule MALT1 inhibitor. Z-VRPR-fmk resulted in a dramatic cell growth inhibition in most of our B-ALL cell lines, with appropriate positive (TMD8) and negative (K562) controls, independent of the cell-of-origin (pro, pre, mature) or the presence of the Philadelphia chromosome. We did not observe a clear correlation between MALT1 level and degree of sensitivity to Z-VRPR-fmk. Interestingly, the two ibrutinib-resistant cell lines RS4;11 and 697, were amongst the top sensitive cell lines to MALT1 inhibition. A similar pattern of cell sensitivity was observed when these cell lines were treated with MI2, resulting in an IC50 at 48h of 0.2 µM in RS4;11 and < 0.5 µM in other sensitive cell lines, which is consistent with published data in sensitive DLBCL cell lines (IC50, 0.2-0.5 µM), and our data on the CLL cell line MEC1 (IC50, 0.2 µM). We then tested freshly collected PBMCs from patients with various blood cancers presenting with a leukemic phase against serial dilutions of MI2 for 48h (7 B-ALL, 24 CLL, and 4 CML). In addition, we included normal B-cells collected from five volunteers. Interestingly, B-ALL samples showed the highest sensitivity to MI2, followed by CLL, while the rest were resistant. The proteolytic activity of MALT1 can be studied by measuring its ability to cleave its targets such as A20, CYLD, BCL10, Roquin, Regnase and RelB. Surprisingly, with the exceptio